The long-term goal of this research project is to characterize the roles of caspase-9 in heart. Apoptosis is a regulated cell deletion process that has been implicated in various cardiovascular diseases. Therefore, specific therapies targeted toward attenuating cardiac apoptosis most likely will have potential therapeutic benefit. Caspase-9, a member of a family of cysteine proteases, plays a critical role in the regulation of apoptosis. In our preliminary study, we showed that-reoxygenation is a strong inducer of apoptosis in adult cardiomyocytes, and reoxygenation-induced apoptosis involves the mitochondria-mediated pathway associated with the activation of caspase-9. This result suggests that caspase-9 is an important regulartor of apoptosis, and that the inhibition of caspase-9 could effectively inhibit cardiomyocyte apoptosis. However, the molecular mechanism of apoptosis in cardiomyocyte is poorly understood. Furthermore, we observed high caspase-9 protein expression in the adult cardiomyocytes, suggesting a possible important role of caspase-9 in adult heart. Yet, very little is known about the role of caspase-9 in heart and the mechanism involved in its activation. Our hypothesis is that caspase-9 is an important reoxygenation-induced apoptosis in adult cardiomvocytes, and the inhibition of caspase-9 will be an effective strategy to attenuate cardiac apoptosis in vitro and in vivo. To test this hypothesis, in Specific Aim 1, we will investigate the role of caspase-9 and its interactions during the induction of cardiac apoptosis. We will study reoxygenation-induced apoptosis in adult cardiomyocytes as our model of cardiac apoptosis. To study the effect of caspase-9 and modulation of its interaction, we will generate and analyze recombinant adenoviruses to overexpress caspase-9 and various mutant forms of caspase-9 in adult cardiomyocytes. In Specific Aim 2, we will examine several strategies to inhibit caspase-9 activity including, pharmacological caspase-9 inhibition, overexpression of the dominant negative form of caspase-9, overexpression of endogenous caspase-9 inhibitor, and overexpression of anti-apoptotic Akt. Finally, in Specific Aim 3, we will define the role of caspase-9 in heart in vivo by generating cardiac-specific caspase-9 knockout mice. Since caspase-9 knock-out by homologous recombination results in perinatal lethality, the effect of caspase-9 in adult tissue can not be studied. Thus, the cardiac specific knockout of caspase-9 using Cre-loxP technology allows us to examine the tissue-specific role of capase-9 in adult animal.